Download 3-D refractive index tomograms and deformability

3-D refractive index tomograms and deformability of individual human red
blood cells from cord blood of newborn infants and maternal blood
HyunJoo Parka,¶, Taegyu Ahnb,¶, Kyoohyun Kima, Sangyun Leea, Songyi Kookb, Dongheon Leeb, In
Bum Suhc, Sunghun Nab,*& YongKeun Parka,*
a
Department of Physics, Korea Advanced Institute of Science and Technology, Daejeon 305-701,
Republic of Korea.
b
Department of Obstetrics and Gynecology, Kangwon National University Hospital, School of Medicine,
Kangwon National University, Chuncheon 200-701, Republic of Korea.
c
Department of Laboratory Medicine, Kangwon National University Hospital, School of Medicine,
Kangwon National University, Chuncheon 200-701, Republic of Korea.
Abstract
Red blood cells (RBCs) from the cord blood of newborn infants have distinctive functions for fetal and
infant development. To systematically investigate the biophysical characteristics of individual cord
RBCs in newborn infants, a comparative study was performed of RBCs from cord blood of newborn
infants, and of adult RBCs from mothers or non-pregnant women, employing optical holographic
micro-tomography. Optical measurements of 3-D refractive index distributions, and of dynamic
membrane fluctuations of individual RBCs, enabled retrieval of the morphological, biochemical, and
mechanical properties of cord, maternal, and adult RBCs at the individual cell level. The volume and
surface area of the cord RBCs were significant larger than those of RBCs from non-pregnant women,
and cord RBCs have more flattened shapes than RBCs in adults. In addition, the Hb content in the cord
RBCs of newborns was significantly greater. The Hb concentration in cord RBCs was higher than for
non-pregnant women or maternal RBCs, but they were within the physiological range of adults.
Interestingly, the amplitude of dynamic membrane fluctuations in cord RBCs were comparable to those
in non-pregnant women and maternal RBCs, suggesting that the deformability of cord RBCs is similar
to that of healthy RBCs in adults.
Keywords: cord blood, maternal, red blood cell, erythrocyte, quantitative phase imaging, optical
imaging
*
Corresponding Authors: [email protected] or [email protected]
¶
These authors contributed equally to this work.
Manuscript information:
Abstract: 197 words
Text: 11 pages, 3970 words
Short running title: 3-D tomograms and deformability of red blood cells from cord blood and maternal
blood
Figures: 4
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1. Introduction
After birth, a newborn infant starts his or her own circulatory system by disconnection of an umbilical
cord that was used for oxygen and nutrient transport from the placenta of the mother. The cord blood
left in the umbilical cord of the newborn infant preserves the characteristics of fetal blood at full term,
and the characterization of individual cord red blood cells (RBCs) of newborn infants is crucial to
understanding maternal-fetal circulation and fetus development, as well as to opening up new
possibilities for diagnosing diseases of newborn infants (1).
Previous studies have revealed that cord RBCs of newborn infants are significantly different from
those in adult blood. For example, macrocytic RBCs, enlarged RBCs with MCV (mean corpuscular
volume) > 110 fL, are predominant in neonatal blood (2), although capillary sizes in newborn infants
are similar to those in adults (3). The cytoplasm of cord RBCs is mainly composed of fetal hemoglobin
(HbF) rather than adult hemoglobin (HbA); HbF exhibits higher oxygen binding affinity than does HbA
(4-6). The life span of cord RBCs (60−80 days) is significantly shorter than normal RBCs (120 days)(7).
The aforementioned characteristics of cord RBCs dramatically change within the first 12 weeks after
birth (8) by producing RBCs with HbA instead of HbF.
Biochemical characterization of cord RBCs and HbF have been extensively studied, but our
understanding of cord RBCs in newborn infants remains incomplete. In particular, the morphological
and mechanical properties of cord RBCs in newborn infants, which are closely related to the
deformability of cord RBCs and cord-blood circulation, have not been fully addressed largely owing to
limitations in measurement techniques. For example, different techniques have led to different
interpretations of the deformability of cord RBCs. Previous work using light scattering or hemolysis
techniques had reported that cord RBCs are more deformable than adult RBCs (9, 10), whereas electron
spin resonance and filtration studies had concluded that there are no significant differences in cell
deformability between cord RBCs and adult RBCs (11-13).
Quantitative phase imaging (QPI), however, circumvents the limitations of previous measurement
techniques and offers unique advantages to measure precisely the morphological and mechanical
properties of cord RBCs. The QPI techniques allow quantitative, non-invasive measurement of optical
phase-delay maps induced by transparent samples (e.g., biological cells and tissues) (14, 15). These QPI
techniques have been utilized for the study of the pathophysiology of RBCs, and provide unique
advantages (16-19). With 3-D QPI techniques, measurement of 3-D refractive index (RI) distributions
of individual RBCs can be carried out, which provide morphological (cell volume, surface area and
sphericity) and biochemical (Hb content and concentration) information about individual RBCs. This
is because RBC cytoplasm is mainly an Hb solution, and the RI of an Hb solution is linearly proportional
to its concentration (20). In addition, dynamic fluctuation in RBC membranes can be precise measured
using QPI techniques, which provides information about the biomechanical properties of the membrane
cortex and the cytoplasm (21-25). Recently, QPI techniques have been utilized for studying the
pathophysiology of RBCs including osmotic changes (26), malaria infection (23, 27, 28), sickle cell
disease (19, 29), and ATP-dependent morphological remodeling (21, 30).
Here, we report optical measurements of the morphological, biochemical and mechanical properties
of cord RBCs in newborn infants. Employing optical holographic micro-tomography, 3-D RI
tomograms and dynamic membrane fluctuation of individual RBCs are non-invasively, and
quantitatively, measured at the individual-cell level. From the measured RI tomograms, morphological
(cell volume, surface area and sphericity) and biochemical (Hb content and concentration) parameters
were retrieved. For a comparative study, optical measurements were performed for RBCs collected from
the blood of non-pregnant women, from the cord blood of full-term newborn infants (within 5 minutes
after delivery), and from maternal blood. The 3-D RI tomographic maps, and the obtained biophysical
parameters, clearly demonstrated the distinctive morphologies of cord RBC: large volumes and more
flattened discocyte shapes, while the maternal RBCs showed elliptical shapes by loss of the dimple. In
addition, the measured dynamic membrane fluctuations showed that cord RBCs have cellular
deformability comparable to RBCs of maternal or non-pregnant women.
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2. Materials and Methods
2.1 Ethics Statement
Human blood studies were conducted according to the principles of the Declaration of Helsinki and
were approved by the responsible ethics committee of Kangwon National University Hospital (IRB
project number: 2012-0128, Chuncheon, Republic of Korea) before start. Human blood were collected
from both non-pregnant women and healthy term pregnant women with 20 years of age or older after
obtaining the written informed consent form which explains the blood can be used for academic research
purposes. Blood of non-pregnant women was obtained via regular medical checkups executed at the
Health Promotion Center of hospital after approval in accordance with the procedures of IRB for the
remaining blood. Maternal blood and cord blood from newborn infant were also collected after
receiving both the written consent directly from both the mother on behalf of herself and her baby and
the verbal consent from the caretakers or the guardians of the minors/children enrolled in the study. This
consent form was also approved by the examined the research plan and consent of the committee on the
IRB. The collecting method for cord blood has minimum risk to the babies or their mothers because
cord blood was collected from the umbilical cord in the placenta after the umbilical cord of the baby
was detached from the placenta.
2.2 Maternal and cord blood collection
Blood samples were collected for healthy term pregnant women in Kangwon National University
Hospital. Cord blood collections were performed by an obstetrician, who attending the delivery. The
umbilical vein of a newborn infant was punctured within 5 minutes after delivery, and 2 mL of cord
blood was collected in anticoagulant agent K2 EDTA (ethylenediaminetetraacetic acid) vacutainer (BD,
NJ,USA). The maternal blood was sampled on the day and non-pregnant women blood was obtained
from remaining blood after examination of health promotion center at Kangwon National University
Hospital. All subjects were healthy without any complications. Total 3 individual of non-pregnant
women as control, and 5 pairs of newborn infants and their mothers were examined. For the optical
measurement, bloods were further diluted 300 times in Dulbecco's PBS buffer (Gibco®, New York,
U.S.A.).
2.3 Holographic Optical micro-tomography
The optical setup of holographic optical micro-tomography is described more detail in previous study
(31). Briefly, the diode-pumped solid state laser (λ = 532 nm, 50 mW, Cobolt, Solna, Sweden) was used
as an illumination source. Using rotating a two-axis galvanometer mirror (GVS012/M, Thorlabs, USA),
the angle of incident beam was varied. For 3-D RI tomography, the optical fields at various incident
illumination angles were measured by employing the common-path laser-interferometric microscopy.
The sample, diluted blood sandwiched between two cover glasses with 25×50 mm (C025501,
MATSUNAMI GLASS Ind., LTD., JAPAN), is placed between the condenser lens (UPLSAPO 60×,
numerical aperture (N.A.) = 0.9, Olympus, Japan) and objective lens (UPLSAPO 60×, N.A. = 1.42,
Olympus, Japan). The second galvanometer mirror reflected the beam from a sample to have same
optical path regardless of incident illumination angle. After the second galvanometer mirror, a
diffraction grating (70 grooves mm-1, #46-067, Edmund Optics Inc., NJ, U.S.A.) spatially split the
scattering beams and then, spatially filtered 0th order beam as a reference was interfered with the 1st
order beam as a sample beam. Then, interferograms were recorded on high-speed sCMOS camera (Neo
sCMOS, ANDOR Inc., Northern Ireland, UK) while the incident beam was scanning spirally with 300
different angles. The total magnification was 240 by an additional 4-f system. From measured optical
fields, 3-D RI distribution of sample was reconstructed using optical diffraction tomography algorithm,
found elsewhere (32).
2.4 Analysis of the red cell parameters
The six red cell parameters are comprised of morphological (cell volume, surface area and sphericity),
chemical (Hb content and Hb concentration), and mechanical (membrane fluctuation) parameters. To
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measure the morphological parameters, we used the reconstructed 3-D RI maps by the diffraction
optical tomography algorithm from measured multiple optical phase maps corresponding to various
illumination angles on the sample. The whole volume of a RBC was calculated by integrating all voxels
inside individual a RBCs. The space corresponding to cytoplasm of a RBC was selected by RI with a
higher value than threshold. The threshold was defined by 50% of RI difference between the maximum
RI of the cell ncell_max and surrounding medium nm for determine the cell boundary, i.e. nthresh = nm + 0.5∙
(ncell_max - nm). Then, the total number of voxels was multiplied by the magnification of the optical system
to translate in a length scale. Next, for surface area measurements, the isosurfaces of individual RBCs
were reconstructed from volume data of 3-D RI maps using MATLAB. Surface area of isosurface was
measured through the sum of the areas of all the patch faces, which were broken down into small
triangular pieces. In addition, the sphericity SI, a dimensionless quantity ranging from 0 to 1, was
obtained by calculating SI = π1/3(6V)2/3/A where the V is the volume and A is the surface area (33, 34).
For the measurement of Hb content, the measured 2-D phase at the normal angle was used. The
Hb content of a RBC was obtained from the integrating 2-D optical phase over entire cell area with RI
increment of proteins and given as
Hb content
=
λ
∑ ∆φ ( x, y ) ,
2πα
(1)
where λ is the wavelength of laser light (532 nm), α is RI increment (0.2 mL/g) (35, 36) and Δφ(x,y) is
2-D optical phase. In addition, the Hb concentration in a RBC was obtained from the Hb content divided
by the cellular volume.
The dynamic membrane fluctuations in RBCs can be quantitatively and precisely measured using
the cDOT. Consecutive dynamic full-field optical phase images of a RBC ∆φ(x, y, t) can be measured
with normal laser illumination, from which dynamic height maps of the RBC can be calculated as, h(x,
y, t) = [λ/(2π⋅∆n)]∆φ(x, y, t), where λ is the wavelength of the illumination laser and ∆n = 〈n(x, y, z)〉 nm is a difference between the mean RI of RBC cytoplasm 〈n(x, y, z)〉 and surrounding buffer medium
nm.
To measure the mechanical parameter, we calculated the dynamic membrane fluctuation from the
successively measured the instantaneous height map h(x,y;t), given as:
h( x,=
y; t )
λ
⋅ ∆φ ( x, y; t )
2π∆n
(2)
The values for the membrane fluctuation were calculated by averaging the root-mean-square of height
displacement over the cell area and given as:
 h( x, y; t ) − hm ( x, y )]2 
∆hrms ( x, y )=
1/2
,
(3)
where hm is the time averaged height at the cell surface.
3. Results
3.1 3-D RI tomograms of individual cord and maternal RBCs
To investigate the 3-D morphological details of individual RBCs, we employed common-path
diffraction optical tomography (cDOT). The cDOT is an optical system for holographic microtomography, which has the capability of measuring the 3-D RI distribution of a sample with high
precision (31). The cDOT measures multiple 2-D optical fields of a sample from different illumination
angles, from which the 3D RI tomogram of the sample n(x, y, z) is reconstructed using a DOT algorithm
(see Material and Methods). Using the cDOT, the 3-D RI distributions of individual RBCs were
measured. Samples were collected from three non-pregnant women (121 RBCs); and from five fullterm newborn infants (215 RBCs) and their mothers (181 RBCs), after delivery. Then the RBCs were
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subjected to quantitative, non-invasive measurement.
The 3-D RI maps of characteristic RBCs from each group are shown in Figs.1A−F. The cord RBCs
from newborn infants are enlarged compared to the RBCs from non-pregnant women. The RBCs from
corresponding mothers, maternal RBCs, are smaller than the cord RBCs, and even smaller than the
RBCs from non-pregnant women. Interestingly, the inner dimple area was absent in maternal RBCs
while the cord RBCs were biconcave discocytes. The characteristic doughnut shapes and loss of
biconcave areas can easily be seen in the rendered isosurfaces of the 3-D RI maps (Figs. 1D−F).
3.2 Quantitative morphological parameters of individual cord and maternal RBCs
For quantitative analysis, we calculated the morphological parameters from the measured 3-D RI
tomograms of the cord and maternal RBCs. These morphological parameters were cellular volume,
surface area, and sphericity (see Material and Methods). The cord RBCs of newborn infants exhibited
cells that were significantly larger than RBCs from non-pregnant women, in terms of both volume and
surface area (Figs. 2A−B). For cord RBCs, the mean values of the cell volume and surface area were
99.5 ± 16.8 fL and 181.8 ± 21.9 μm2; whereas those of the RBCs from mothers and non-pregnant women
were 89.6 ± 8.0 fL and 139.2 ± 17.4 μm2, and 87.2 ± 13.0 fL and 148.9 ± 16.5 μm2, respectively. The
volumes of RBCs from non-pregnant women measured using cDOT, were consistent with physiological
range and the mean cell volumes (MCV), which were independently measured using automated bloodcell counters, based on the complete blood count (CBC). These are indicated by gray lines in Fig. 1.
Note that the surface area cannot be obtained from CBC measurements. The mean corpuscular volume
with RDW (red cell distribution width) measured by the CBC were 87.3 ± 15.3, 99.2 ± 14.4, and 89.6
± 12.2 fL for non-pregnant adult RBCs, cord RBCs and maternal RBCs, respectively. The volume and
surface area of maternal RBCs were comparable to those of the RBCs from non-pregnant women (Fig.
2C). The mean values of the cell volume and surface area were 89.4 ± 16.4 fL and 139.2 ± 17.4 μm2.
The increased volume in cord RBCs is consistent with previous CBC measurements (2).
In order to analyze the degree of biconcave shape in the cord and maternal RBCs quantitatively,
we calculated the sphericity from the measured cell volume and surface area. The sphericity is a
dimensionless measure of how spherical an object is. The sphericity of a perfect sphere is ‘1’ and that
of a flat surface is ‘0’. The mean sphericity value of the RBCs from non-pregnant women, exhibiting a
characteristic biconcave shape, was 0.64 ± 0.06. The sphericity of cord RBCs and maternal RBCs were
0.57 ± 0.07, and 0.70 ± 0.09, respectively. This indicates that in comparison with healthy RBCs from
non-pregnant women, the enlarged cord RBCs exhibit more flattened shapes, and the maternal RBCs
are more spherical.
3.3 Cellular Hb content and concentration in individual cord and maternal RBCs
To quantify the biochemical characteristics of RBC cytoplasm, Hb content and concentration in the cord
and maternal RBCs were quantified from the measured 3-D RI maps. The Hb content of individual
RBCs was retrieved from the measured 2-D optical field of cells using the cDOT. Then, the Hb
concentration of individual RBCs could be calculated from the Hb content and cell volume of individual
RBCs (see Materials and Methods).
The mean values of Hb content were 29.5 ± 4.7, 35.9 ± 6.9, and 30.3 ± 5.2 pg for non-pregnant
adult RBCs, cord RBCs, and maternal RBCs, respectively (Fig. 3A). The Hb content in cord RBCs was
22% greater than that of RBCs from non-pregnant women, while the Hb content of the maternal RBCs
was comparable to that of the RBCs from non-pregnant women. This result, obtained with the cDOT,
is also consistent with the CBC measurements: mean corpuscular hemoglobin values were 29.6 ± 1.2,
34.7 ± 0.6, and 30.7 ± 3.9 pg for the non-pregnant adult RBCs, cord RBCs, and maternal RBCs,
respectively (gray dotted lines in Fig. 3A).
The mean values of Hb concentration were 33.8 ± 2.6, 36.3 ± 4.7, and 34.2 ± 3.3 g/dL for nonpregnant adult RBCs, cord RBCs, and maternal RBCs, respectively (Fig. 3B). The mean value of Hb
concentration of the cord RBCs was measurably greater than that of RBCs from non-pregnant women,
and of maternal RBCs.
5
As shown in Fig. 3C, the correlations between Hb content and cellular volume were positive in all
three RBC groups. The linear slope in the correlation map indicated the Hb concentration of each RBC
group, and the values of slope were 0.334 ± 0.005, 0.360 ± 0.06, and 0.337 ± 0.004 pg/fL for the RBCs
from non-pregnant women, cord RBCs, and maternal RBCs, respectively. These results are also in good
agreement with the CBC measurements: mean corpuscular Hb concentration was found to be 33.9, 34.6,
and 33.34 g/dL for the RBCs from non-pregnant women, cord RBCs and maternal RBCs, respectively
(gray dotted lines in Fig. 3B). Although the Hb concentration in cord RBCs were slightly greater, all
the mean values of Hb concentration were within the reference range of a healthy adult (33–36 g/dL).
3.4 Cellular elasticity of individual cord and maternal RBCs
To investigate the mechanical properties or deformability of individual cord and maternal RBCs, the
dynamic membrane fluctuations of the RBCs were measured. Due to the soft and elastic properties of
membrane cortex structures, RBCs exhibit dynamic membrane fluctuations driven by thermal or
metabolic energy (21, 37-41). The dynamic membrane fluctuation manifests the deformability of RBC
membranes, which is strongly correlated with the structures of the lipid membrane and spectrin network,
and alternations caused by various pathophysiological conditions (17, 19, 32).
The dynamic membrane fluctuations in RBCs were quantitatively and precisely measured using the
cDOT. Consecutive dynamic full-field optical phase images of individual RBCs were measured with
normal laser illumination, from which the mean and dynamic height maps of RBCs were calculated
(see Materials and Methods). The representative mean and dynamic height maps of individual RBCs
in each group are presented in Figs. 4A-C. Consistent with the results of the 3-D rendered isosurfaces
(Figs. 1D-F), the mean-cell-shape results demonstrate that the maternal RBCs exhibit spherical shapes
without center-dimpled regions, whereas the characteristic donut shapes occur in the cord RBCs as well
as in the RBCs from non-pregnant women. The instantaneous displacement maps of the dynamic
membrane fluctuations are shown in Fig. 4D-F.
In order to quantify the deformability of individual RBCs, the membrane fluctuations were
calculated as the spatially averaged root-mean-squared (RMS) height displacement (Fig. 4G). The
representative membrane fluctuations of an RBC from a non-pregnant adult, cord blood and maternal
blood are presented in Figs. 4D-F, respectively. The RMS height displacements of the cord RBC are
homogenous over the cell area, and compatible with those of non-pregnant adult RBC and maternal
RBC. The mean values of membrane fluctuation were 52.7 ± 5.8, 50.9 ± 5.9, and 52.7 ± 7.1 nm for
RBCs from non-pregnant women, the cord, and maternal RBCs, respectively. There were no statistical
differences in membrane fluctuation among the three groups of RBCs, indicating that cellular
deformability of the cord RBCs and the other RBCs are not significantly altered, despite considerable
differences in cell volume and surface area.
4 . Discussion
We presented the measurements of morphological, biochemical and mechanical characteristics of
individual cord RBCs and maternal RBCs. Quantitative, non-invasive measurements of RBCs using the
cDOT, 3-D RI maps and dynamic membrane fluctuations were made. From these, the following
important red cell parameters were retrieved systematically: cell volume, surface area, sphericity, Hb
content, Hb concentration, and membrane fluctuation. The measured values of cell volume, Hb content,
and Hb concentration were consistent with independent CBC measurements, and also consistent with
previous reports (7). To date, this is the first reported experimental measurement of surface area,
sphericity, and membrane fluctuation of cord and matching maternal RBCs.
We should note that the cDOT measurements provide more detailed information about individual
RBCs from cord blood, compared to existing CBC blood tests. For example, the cDOT provides
visualization of structural details of the RBC (e.g., 3D shape, surface area, and sphericity), whereas the
CBC measurement only provides cell sizes from impedance measurements. The cDOT also provides
chemical information about individual RBCs, whereas the CBC test measures ensemble averaged
information. Furthermore, deformability measurements are not available using CBC blood tests.
6
Our results using the cDOT clearly show that the cord RBCs of full-term newborn infants exhibit
significant different morphology from the RBCs of non-pregnant women. The volume and surface area
of the cord RBCs were 14% and 30% larger, respectively, than those of RBCs from non-pregnant
women. The sphericity of the cord RBCs was 11% less than that of the RBCs from non-pregnant women,
indicating that cord RBCs have more flattened shapes. The Hb content in the cord RBCs of newborns
was significantly greater: the Hb content of the cord RBCs was 22% and 18% greater than that of nonpregnant adult or maternal RBCs. In addition, the Hb concentration in cord RBCs was higher than for
non-pregnant women or maternal RBCs, but they were within the physiological range of adults.
Interestingly, the amplitude of dynamic membrane fluctuations in cord RBCs were comparable to those
in non-pregnant women and maternal RBCs, suggesting that the deformability of cord RBCs is similar
to that of healthy RBCs in adults.
It is speculated that these differences in cord RBCs might have resulted from evolution in order to
meet the demand for high oxygen consumption by the fetus. Enlarged cell volume with higher Hb
concentration in the cord RBCs could carry oxygen to fetal tissues more efficiently. Because the high
oxygen-binding affinity of HbF in cord RBCs facilitates the transport of oxygen between two different
circulatory systems via an umbilical cord, unique morphological and biochemical properties of cord
blood enhance oxygen transport from placenta to fetus.
The question then arises whether this remodeling of cord RBCs is beneficial or detrimental to fetal
blood circulation. Although this question is not directly accessible to current experimental study, our
measurements of membrane fluctuations suggest that cord RBCs may not be significantly different from
other healthy RBCs in their ability to pass through small capillaries and restore their original shapes.
This is based on their remarkably soft and elastic properties. Despite the enlarged cell volumes in cord
RBCs, their decreased sphericity indicates more discocytic shapes than exhibited by healthy RBCs in
adults, suggesting that cord RBCs may still have good ability in passing through narrow passages.
The measured dynamic membrane fluctuations of the cord RBCs, the maternal RBCs, and the nonpregnant adult RBCs are not significantly different from one another. This result is consistent with a
previous study using osmotic frangibility which reports indistinguishable cellular deformability
between full-term newborn infants and adults RBCs (9). However, considering that other macrocytic
RBCs produced due to various clinical situations, including anemia and chronic alcoholism (42, 43),
exhibited more deformable RBCs, it is intriguing that the cord RBCs with enlarged cell volumes exhibit
dynamic membrane fluctuations comparable to healthy RBCs. In addition, the maternal RBCs exhibit
loss of characteristic dimple shapes and became sphere-like. However, the loss of dimple shapes found
in ATP-depleted RBCs accompanied decreases in membrane fluctuations, which was not the case in the
measured maternal RBCs. Taken together, our results imply complex remodeling in membrane cortex
structures in cord RBCs and maternal RBCs.
We presented the optical measurements of morphological, biochemical and mechanical properties
of individual cord RBCs and performed comprehensive comparative analysis of maternal RBCs and
non-pregnant healthy RBCs. The present method will open possibilities for diagnosis of diseases of
newborn infants and their mothers, as well as for the study of pathophysiology of cord RBCs and their
implications in fetal circulation. From the technical point of view, the use of the quantitative phase
imaging unit can convert an existing microscope into a quantitative phase microscopy (44, 45), and will
further expand the applicability of the present technique.
ACKNOWLEDGEMENTS
This work was supported by from 2014 Kangwon National University Hospital Grant, KAIST-Khalifar
University Project, APCTP, and National Research Foundation (NRF) of Korea (2012R1A1A1009082,
2012-M3C1A1-048860, 2013R1A1A3011886, 2013M3C1A3063046, 2013K1A3A1A09076135,
2014M3C1A3052537, 2014K1A3A1A09063027).
AUTHOR CONTRIBUTIONS
7
Y.P and S.N. developed the experimental idea. H.P. performed the optical experiments and analyzed the
data. S.N. prepared the sample of maternal and cord blood. S.N. and Y.P. conceived and supervised the
study. All authors discussed the experimental results and wrote the manuscript.
COMPETING FINALCIAL INTERESTS
The authors declare no competing financial interests.
8
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Figures with legends
Fig. 1 A-C, Reconstructed 3-D RI tomograms of RBCs from a healthy non-pregnant women (A), a RBC
from cord blood (B), and from a mother (C), respectively. The cross-sectional slices of the RI
tomograms are shown in the x-y (top left panel), y-z (right panel), and x-z (bottom panel) planes. D−F,
3-D rendered isosurface images of the RBCs in A−C.
Fig. 2 Red cell indices for non-pregnant women RBCs (N = 121, black circles), cord RBCs from fullterm newborn infants (N = 215, red circles), and their maternal RBCs (N = 181, blue circles): RBCs
cellular volume (A), RBCs surface area (B) and sphericity index (C). Each symbol represents an
individual RBC measurement and the horizontal solid line is the mean value, and the vertical lines, STD
error bars. Gray dot lines in (A) correspond to averaged MCV from the relevant CBC blood test, with
vertical lines of RDW (red cell distribution width). The symbol * indicates a p-value <0.005.
11
Fig. 3 RBC dry mass (A), and Hb concentration (B), of non-pregnant women RBCs (N = 121, black
circles), fetal cord RBCs (N = 215, red circles) and maternal RBCs (N = 181, blue circles). Each symbol
represents an individual RBC measurement and the horizontal line is the mean value, vertical lines
indicate STD error bars. Horizontal gray dot lines in (A) and (B) correspond to averaged MCH and
MCHC from the CBC blood test. C, Correlation map of RBC cellular volume and RBC dry mass with
fitted linear slopes for cord RBCs (red solid line) and maternal RBCs (blue dashed line). The symbol *
indicates a p-value <0.005.
Fig. 4 (A-C) Representative 2-D topographic images of RBCs from the non-pregnant women, the cord
blood of a newborn infant and a mother, respectively. (D-F) Their corresponding dynamic membrane
fluctuations. The color bar scales are in μm (top row) and nm (bottom), respectively. (G) The averaged
membrane fluctuations of individual RBCs in each group: non-pregnant women (black circles), cord
blood of full-term newborn infants (red circles) and their mothers (blue circles). The horizontal solid
line is the mean value; the vertical lines are STD error bars.
12